APPLIED BIOLOGICAL SCIENCES Correction for “Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity,” by Carl W. Fuller, Pius S. Padayatti, Hadi Abderrahim, Lisa Adamiak, Nolan Alagar, Nagaraj Ananthapadmanabhan, Jihye Baek, Sarat Chinni, Chulmin Choi, Kevin J. Delaney, Rich Dubielzig, Julie Frkanec, Chris Garcia, Calvin Gardner, Daniel Gebhardt, Tim Geiser, Zachariah Gutierrez, Drew A. Hall, Andrew P. Hodges, Guangyuan Hou, Sonal Jain, Teresa Jones, Raymond Lobaton, Zsolt Majzik, Allen Marte, Prateek Mohan, Paul Mola II, Paul Mudondo, James Mullinix, Thuan Nguyen, Frederick Ollinger, Sarah Orr, Yuxuan Ouyang, Paul Pan, Namseok Park, David Porras, Keshav Prabhu, Cassandra Reese, Travers Ruel, Trevor Sauerbrey, Jaymie R. Sawyer, Prem Sinha, Jacky Tu, A. G. Venkatesh, Sushmitha VijayKumar, Le Zheng, Sungho Jin, James M. Tour, George M. Church, Paul W. Mola, and Barry Merriman, which published January 24, 2022; 10.1073/pnas.2112812119 (Proc. Natl. Acad. Sci. U.S.A. 119, e2112812119).
The competing interest statement has been revised to add: “D.A.H., S. Jin, J.M.T., and G.M.C. are members of the Roswell Biotechnologies Inc. Scientific Advisory Board, which entitles them to certain financial interests in the company. D.A.H. has also performed paid consulting for Roswell.”
The affiliation for Drew A. Hall should not include Roswell Biotechnologies Inc., San Diego, CA 92121.
The authors note that Fig. 3 appeared incorrectly. Fig. 3F was missing several text labels and Fig. 3 E and H each had label formatting errors. The corrected figure and its legend appear below.
Fig. 3.
A survey of diverse molecular electronic sensors for binding and enzyme activity, shown to scale in molecular renderings, along with corresponding summary experimental results. (A–D) Protein and small-molecule binding kinetics. As a model system for showing protein binding and small-molecule binding, this sensor is configured to observe (A) a DNA polymerase binding a primer/template, and (C) a nucleotide binding into the polymerase pocket. For this model system, a 17-mer ssDNA template is conjugated to the peptide bridge at its 5′ end (and with 3′ end blocked to prevent the polymerase from binding that site). A complementary 14-mer primer strand is then bound to this, on the distal end of the 17-mer, to create a primer site on the sensor with the 3′-OH available for polymerase binding. (B) Summary kinetics (dwell time, fraction of time bound) for Klenow DNA polymerase binding to the primer site, as polymerase concentration is titrated from 0.008 to 3.8 µM, in a background of 100 nM 14-mer primer to suppress primer dissociation. The inferred binding affinity of the polymerase is Kd = 530 nM. (D) A nucleotide titration is performed to observe the binding in the polymerase pocket, in a noncatalytic buffer so as to observe the binding kinetics without incorporation. A 45-mer template is on the bridge, and a 31-mer primer was bound to the distal end of the template, so that the first template base (A) is complementary to the nucleotide being tested (T). The nucleotide was added in concentrations of 2.5, 5, and 15 µM along with the 100 nM polymerase and primer in the presence of a buffer that has 10 mM Sr2+ (without Mg2+), in which nucleotide incorporation cannot occur. In this buffer, the dNTP will repeatedly bind and dissociate from the polymerase pocket, and the resulting summary binding kinetics are shown. This also serves to illustrate the detection of small molecule binding. (E and F) Aptamer sensors: Aptamer sensors were constructed, here targeting the SARS-CoV-2 S protein with a 57-mer DNA aptamer (E) and targeting the SARS-CoV-2 N protein with a 97-mer DNA aptamer (SI Appendix, Fig. S7B-2), both taken from the literature. (F) The concentration response titration curves for both the S aptamer and N aptamer sensors, for a range of applied target protein concentrations. The binding affinities, Kd, derived from these curves (6.4 nM, 39 nM) are similar to those reported for standard bulk aptamer binding assays in solution. (G and H) Antibody–antigen sensors. As a model system, a fluorescein–antifluorescein antigen–antibody pair was used, with the fluorescein antigen presented on the sensor by tethering it to the bridge using a ssDNA oligo as a linker. A 45-mer oligonucleotide was used, with the 3′ (distal) end of the DNA capped with a fluorescein during synthesis. A commercial antifluorescein antibody (Fab) was added in TKS buffer on the chip. The summary kinetics are shown for dwell time and fraction of time bound, as the concentration of antibody is titrated over the range shown. The inferred binding affinity was Kd = 1.3 µM. It was observed that all binding signals were extinguished when 4 mM of free fluorescein was added to saturate the antibody, verifying the specificity of the binding signal. (I and J) A CRISPR/Cas enzyme activity sensor. To assemble a Cas enzyme as a probe on the bridge, first a guide RNA targeting a dsDNA target for a CRISPR/Cas12a enzyme was conjugated to the bridge, and these were assembled on chip. A Cas12a enzyme was provided in solution and allowed to dock to the guide RNAs on the bridges, thereby programming it for the target dsDNA, and also effectively tethering it to the bridge as a probe. For these experiments, the guide RNA is a 40-mer, attached to the bridge peptide using click chemistry at the 13th nucleotide, which is the base that extends furthest outside the enzyme in the pseudoknot loop. The kinetics are summarized in the titration curve, showing fraction of time bound saturating as the dsDNA target varies in concentration, in the presence of a concentration of 20 nM free (untargeted) Cas12a enzyme. Thus, this configuration acts directly as a sensor for the dsDNA target, without assessing posttarget-binding nonspecific single-stranded nuclease activity. This latter nonspecific activity is also observable on the sensor when provided with a ssDNA substrate. The observed binding affinity for the dsDNA target is Kd = 3 pM. The experimental buffer was 20 mM Tris·HCl pH 8.0, 20 mM KCl, 10 mM SrCl2, 4 mM DTT. Two examples of raw sirgnal traces for D (small molecule) and E (protein) are included in SI Appendix, Fig. S7 B and F, showing that the character of the bridge current signals is similar for these diverse probes.
The authors note that, in the SI Appendix, page 21, the figures referenced in the first full paragraph, lines 1–10, appeared incorrectly. Additional minor edits have been made throughout the SI Appendix for clarity and it has been corrected online.
An additional SI Appendix has been added disclosing patent information filed by Roswell Biotechnologies, Inc.